Gene fusions will be constructed in vitro that join the lacZ gene coding for Beta-galactosidase to the ilvG, ilvE, ilvD, and ilvA genes of the ilvGEDA cluster. In addition, internal segments of the ilvGEDA region will be subcloned to promoter and terminator testor plasmids. Both wild type and mutant ilv DNAs will be used for these fusions. This will make it possible to monitor and quantitatively compare the activities of promoters, terminators and attenuator(s) located within and upstream of the ilvGEDA cluster. These plasmids will be transferred to isogenic host strains with and without specific mutations, including rho120, leuS31, ilvG671(ValR) and ilvA538, that are known to affect the expression of the ilvGEDA gene cluster. The effects of these chromosomal mutations upon the expression of each gene will be determined by growth in the presence of an excess of end products and with separate limitation for leucine, isoleucine, and valine. Specific ilv-lac fusions will be moved to the chromosome by homologous recombination to test for effects of copy number that may be due to a limiting concentration of an effector molecule. Some ilv-lac fusions will be used to select for mutations that affect these internal sites of transcription initiation and termination, and these mutations will be characterized by direct DNA sequence analysis. The presence of internal sites of transcription initiation and termination in vivo will be assessed by Northern hybridization to mRNA extracted from cells grown with excess or limiting end products. The 5' and 3' termini of these mRNA species will be precisely identified by nuclease S1 digestion of DNA-RNA hybrids. In vitro transciption will be used to analyze interal control sites postulated to precede the ilvE and ilvD genes. The presence of stem and loop structures that we have detected in the ilvGEDA region will be analyzed by nuclease S1 treatment of supercoiled plamids. An in vitro transciption and translation system primed with ilv and ilv-lac templates will be used to confirm the in vivo results and to distinguish between control of transcription and translation using coupled and uncoupled systems.